DE3330573A1 - METHOD FOR PRODUCING IMMOBILIZED 1 OR MORE NUCLEOPHILE GROUP (S) COMPOUNDS AND ACTIVATED POLYMERS CARRIER - Google Patents

Description

333057:

DR. STEPHAN G. BESZgDES ^; -PATENT ADVOCATE

VMR: 101265

APPROVED REPRESENTATIVE ALSO AT THE EUROPEAN PATENT OFFICE

PROFESSIONAL REPRESENTATIVE ALSO BEFORE THE EUROPEAN PATENT OFFICE

D-ACHAU NEAR MÖNCHEN

BOX 1168

MONCHENER STRASSE 8OA Federal Republic of Germany

TELEPHONE: D AC H AU 081 31/4371 TELEX: 527537 bepat d

Postal checking account Munich (BLZ 700 100 80)

Account No. 1 368 71-801 Bank account No. 906 370 at the Sparkasse Dachau

(BU 700 515 40)

(VIA Bayerische Landesbank

Girozentrale. Munich)

P 1 793

Claims and Description

for patent application

EEAJSAL FHTOMVEGXSZERGYAJR

Budapest, Hungary

concerning

Process for the production of immobilized 1 or more nucleophilic group (s) Compounds and activated polymeric carriers

«= ■■

description

The invention "relates to a method of manufacture of immobilized 1 or more nucleophilic group (s) having Compounds and activated polymeric carriers suitable for immobilizing 1 or more nucleophiles Group (s) containing compound (s), for immobilizing 1 or more nucleophilic group (s) containing compounds, each, for example, amino acids, peptides, proteins and carbohydrates.

'In the past few years, several methods of immobilization have been used of small molecular weight compounds and biopolymers, for example proteins, on a solid Carriers worked out (Zaborsky, 0. E.: Immobilized Enzymes [West E. C, ed.] [1973], CRC Press, 'Cleveland Ohio; Chibata, I .: Immobilized Enzymes [Chibata I., ed.} [1978], Pages 9 to 107, Kodansha Ltd., Tokyo, Goldstein L., Manecke G .: Applied Biochemistry and Bioengineering {Wingard, L. B., Katchalski-Katzir, E. and Goldstein, L., ed.] [1-979], Volume 1, pages 23 to 126, Academic Press, Ine., Uew York).

The general disadvantages of the known methods are summarized as follows:

A) Special reaction conditions are required.

B) In some methods, the bond between the support and the ligand is achieved using special or difficult to access coupling reagents formed.

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C) Some used. Coupling reagents, for example thiophosgene, are extremely toxic and make . the application of separate security measures, which are associated with great effort, is required.

·. The object of the invention is to provide a method while eliminating the disadvantages of the prior art "for the preparation of immobilized compounds containing 1 or more nucleophilic group (s) and activated polymers Carriers suitable for immobilizing 1 or more nucleophilic group (s) containing compounds by which the Immobilize with a covalent bond under simple conditions, by means of less toxic and easy conditions accessible activating agent and with easily accessible, advantageous properties that can also be produced on an industrial scale having carriers can be carried out, too create.

The above has surprisingly been achieved by the invention.

The invention is based on the surprising finding that the amide groups, especially carboxamide groups, have Polymers with quinones with activation of their. Amide groups, interact and the so formed activated polymers for the binding of compounds containing nucleophilic groups by covalent bonds in advantageous Way are capable. This plague position is all the more unexpected as the carboxamides are only known to be have poor responsiveness and due to the knowledge disclosed in the literature, the skilled person could not draw the conclusion that they would react without quinones.

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The invention therefore relates to a process for the production of immobilized compounds having 1 or more nucleophilic group (s) by activating polymeric carriers used in buffer solutions with activating or coupling agents used in solution and treating the compounds having 1 or more nucleophilic group (s) with the polymeric carriers activated in this way and using separation and washing operations, which is characterized in that the polymeric carrier is a polymer containing amide groups in a buffer solution with a pH of 3 to 11 that is free from amino, sulfhydryl and / or substances containing hydroxyl groups is swollen, then the solution of a quinone in a water-miscible organic solvent is added in such an amount that the final concentration of the quinone is 2 to 100 Mi THm r / l / i, then the mixture Is activated for 0.5 to 48 hours at 273 to 34-3 ° £, then the unreacted quinone is removed by washing with a mixture of water and a water-miscible organic solvent and then with distilled water and / or a buffer solution, then the resulting gel of the activated '. Amide group-containing polymer a 0.001 to 1.0 M buffer solution with a "pH value of 3 to 11 of the 1 or more nucleophilic group (s) having low molecular weight compound (s) or a buffer solution of the 1 or more nucleophilic group (s) containing macromolecular compound (s) with a pH of 3 to 11 at a concentration of 3 to 100 mg / ml is added, the suspension obtained is incubated at 260 to 313 ° K for 20 to 30 hours and then the gel is separated off and the non-bonded compound (s) having 1 or more nucleophilic group (s) is separated or grounded by washing.

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Any polymers containing amide groups can be used lind "any buffer solutions, in the latter case provided the Condition is met that they do not contain any substances with amino, Containing sulfhydryl and / or hydroxyl groups, is used will.

The term "polymers" also includes copolymers. Acrylamide polymers or copolymers such as acrylamide / ΪΤ, ΙίΤ ¹ - -methylene-bis (acrylamide) copolymers can advantageously be used as polymers containing amide groups.

Preferably is or are used as buffer solution (s) for the amide group-containing polymer and / or the compound (s) having 1 or more nucleophilic group (s) with a low molecular weight or macromolecular compound (s) [a] those with a pH 6 to 8 used.

A potassium phosphate buffer solution can advantageously be used as the buffer solution for the polymer having amide groups will.

Can be advantageous as a buffer solution for the 1 or more Compound (s) having nucleophilic group (s) with low molecular weight or macromolecular compound (s) a potassium / sodium phosphate buffer solution, Sodium formate buffer solution or sodium carbonate / entrium bicarbonate buffer solution can be used.

It is also preferred to use p-benzoquinone as the quinone.

As a solvent in which the quinone is used, may or may be advantageous, if appropriate

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aqueous, alkenols with 1 to 3 carbon atoms or, optionally aqueous, dioxane or mixtures of such can be used.

Furthermore, it is preferred to use the quinone solution in such an amount that its final concentration 4-0 to 60 millimoles / l.

Furthermore, it is preferred to activate the mixture from the buffer solution of the amide group serving as a carrier having polymer and the solution of the quinones at 310 to be carried out up to 33O ° K.

In addition, it is preferred to carry out the activation of the mixture of the buffer solution of the polymer having amide groups serving as a carrier and the solution of the quinone for 20 to 30 hours, particularly 2A hours.

Advantageously, for washing the polymer having activated amide groups and serving as a carrier, it can be used as a mixture of water and a water-miscible organic solvent, aqueous dioxane, in particular 20% by volume aqueous dioxane can be used.

It is advantageous to use a buffer solution for washing the polymer having activated amide groups and serving as a carrier a phosphate buffer solution can be used.

The carrier having activated amide groups and serving as carrier can be in the moist state or lyophilized be stored.

Appropriately, the pH of the solution becomes 1 or more compound (s) having nucleophilic group (s) with low molecular weight or macromolecular compound (s) according to their stability within the above

solidified area chosen.

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It is advantageous in the case of 1 or more nucleophiles Low molecular weight compound (s) comprising group (s) such a solution of the same with a concentration of 0.005 "to 0.05 mol / l, in particular 0.01 mol / l, used.

. In the case of 1 or more nucleophilic group (s) having macromolecular compound (s), one is advantageous such a solution thereof at a concentration of 10 to 30 mg / ml, particularly 20 mg / ml, is used.

The incubation of the suspension obtained of the compounds having 1 or more nucleophilic group (s) is preferably carried out. with · low molecular weight respectively. macromolecular compound (s) and that serving as a carrier activated polymer containing amide groups at 260 to 280 ° K carried out.

It is also preferred to incubate the resulting suspension the low molecular weight compound (s) having 1 or more nucleophilic group (s), respectively macromolecular compound (s) and the polymer having activated amide groups serving as a carrier 22 to 26 hours, especially 24 hours, to be carried out for a long time.

Is advantageous for washing the gel obtained immobilized compound (s) having 1 or more nucleophilic group (s) one, optionally sodium chloride, - · buffer solution containing preferably in an amount of 0.8 to 1.2 mol / l, in particular 1.0 mol / l, preferably Sodium acetate buffer solution, especially with a sodium acetate concentration from 0.05 to 0.15 mol / l, very particularly 0.1 mol / l, is used. It is also advantageous to follow this up with a sodium bicarbonate solution, preferably with a concentration of 0.05 t> is 0.15 Hol / l, in particular

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The invention also relates to activated polymers Carrier suitable for immobilizing 1 or more nucleophilic group (s) having compound (s), which through Activation of polymers used in buffer solutions Carriers with activating or coupling agents used in solution and with the use of separating agents and Washing operations have been obtained and characterized are that for their production as a polymeric carrier, a polymer containing amide groups in a buffer solution with a pH value of 3 to 11, which are free from substances containing amino, sulfhydryl and / or hydroxyl groups is, has been swollen, then the solution of a quinone in a water-miscible organic solvent in such an amount that the final concentration of the quin ο nes 2 to 100 millimoles / 1 has been added, then the mixture for 0.5 to 48 hours at 273 bis 34-3 ° K activated and then the unreacted Quinone by washing with a mixture of water and a water-miscible organic solvent and then removed with distilled water and / or a buffer solution.

The above preferences apply insofar as they facilitate the production of the activated 1 or more serving as a carrier Amide group (s) containing polymers themselves concern, here too.

The invention has the following advantages in particular:

a) The bond formed is stable.

b) Has the method according to the invention can under

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Turning of easily accessible substances carried out will.

c) The method according to the invention is to be used ■■ ·. ".with a wide range of different carriers suitable.

. d) The method according to the invention and the invention ; according to activated polymeric carriers can be used in a wide pH range.

The invention is explained in more detail with reference to the following examples.

example 1

0.1 g of a xerogel of an acrylamide / ΪΓ, ΪΓ- -methylene-bis (acrylamide) copolymer [Akrilex P-100 Xerogel, manufacturer: Reanal Finomvegyszergyär, Budapest, Hungary] in 4.0 ml of a 0.1 m Sörensen buffer solution (pH = 0) suspended, whereupon 1.0 ml of a 0.25 M reading of p-Benzochirxn in 20 vol .-% - aqueous dioxane was added and the suspension was kept at 32J ⁰ K for 24 hours . The swollen gel was separated and washed successively with 70 ml of 20% by volume aqueous dioxane and 70 ml of distilled water.

The activated gel was filtered through a vacuum filter and 2.0 ml of a 0.01 M solution of cf-aminobutyric acid in a 0.1 M potassium / sodium phosphate buffer solution (pH = 7 * 5) was added to it . The suspension was incubated for 24 hours at 277 ° K, the gel was separated and the unbound ₍ γ-aminobutyric acid was removed by successive washing with a 1.0 mol / l

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Sodium chloride containing 0.1 ι sodium acetate buffer solution (pH = 5.0) and a 0.1 M sodium bicarbonate solution (pH = 9.5) removed- The amount of bound ^ -amino- "butyric acid was 4, 0 micromoles, which corresponds to a yield of 20% based on that introduced into the reaction mixture ν-aminobutyric acid amount, corresponds.

Example 2

0.1 g of a xerogel of an acrylamide / UjIT ¹ - -methylene-bis- (acrylamide) copolymer [Akrilex P-100 Xerogel, manufactured by Reanal Einomvegyszergyär, Budapest, Hungary] in 4.0 ml of a 0.1 m Sörensen Buffer solution (pH = 8.0) 'suspended, whereupon 1.0 ml of a 0.25 M solution of p-benzoquinone in 20% by volume dioxane was added and the suspension was left to stand at 323 ° K for 24 hours became. The swollen gel was separated and washed successively with 70 ml of 20% by volume aqueous dioxane and 70 ml of distilled water.

The activated gel was filtered through a vacuum filter and 2.0 ml of a solution of hind serum albumin having a concentration of 20 mg / ml in a 0.1 M sodium formate buffer solution (pH = 3.0) was added to it. The suspension was incubated for 24 hours bei'277 K ⁰ ".. The gel was separated off and the bovine serum albumin was not bound by washing successively with a 1.0 Get / l sodium chloride containing 0.1 m ITatriumac etatpuff redemption (pH-V / ert = 5 »0) and a 0.1 M sodium bicarbonate solution (pH = 8.5) removed. The amount of bound bovine serum albumin was 10 mg, which corresponds to a yield of 25%, based on the bovine serum albumin added to the reaction mixture. is equivalent to.

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Example 3

It was 0.1 g of a xerogel of an acrylamide / Ιϊ, ϊΓ- -Methylene-bis- (acrylamide) copolymers CAkrilex P-100 Xerogel, Manufacturer: Reanal Finomvegyszergyar, Budapest, Hungary] in 4.0 ml of a 0.1 M Sörensen buffer solution (pH = = 8.0) suspended, before 1.0 ml of a 0.25 M solution of p-Benzoquinone in 20% by volume aqueous dioxane was added and the suspension was kept at 323 ° K for 24 hours became. The swollen gel was separated and sequentially mixed with 70 ml of 20% by volume aqueous dioxane and 70 ml washed in distilled water.

The activated gel was filtered through a vacuum filter and given 2.0 ml of a concentration of 20 mg / ml solution of bovine serum albumin in a 0.1 M potassium / sodium phosphate buffer solution (pH value = = 6.0) added. The suspension was "at" for 24 hours 277 E ". The gel was separated and the non-bound Bovine serum albumin was obtained by sequential Wash with a 1.0 mol / l sodium chloride containing 0.1 M Sodium acetate buffer solution (pH value = 5 * 0) and a 0.1 M sodium bicarbonate solution (pH = 8.5). The amount of bound bovine serum albumin was 4.5 mg, which corresponds to a yield of 11.25% based on the amount of bovine serum albumin introduced into the reaction mixture, is equivalent to.

Example 4

It was 0.1 g of a Xerogelcs of an acrylamide / ΙΤ, ΙΓ ¹ -methylene bis (acrylnmid) copolymer r, [Akrilox P-100 Xerogel,

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Manufacturer: Reanal Finomvegyszergyar, Budapest, Hungary] in 4.0 ml of a 0.1 M Sörensen buffer solution (pH = 8.0) suspended, whereupon 1.0 ml of a 0.25 M solution of p-benzoquinone in 20% by volume aqueous dioxane was added and the suspension was kept at 323 ° K for 24 hours. The swollen Gel was separated and sequentially with 70 ml of 20% by volume aqueous dioxane and 70 ml of distilled Water washed.

The activated gel was filtered through a vacuum filter and 2.0 ml of a solution of bovine serum albumin having a concentration of 20 mg / ml in a 0.1 M sodium carbonate / sodium bicarbonate buffer solution (pH = 9 »0) was added to it. The suspension was incubated at 277 ° K for 24 hours. The gel was separated and the unbound bovine serum albumin was washed successively with a 0.1 M sodium acetate buffer solution (pH = 5 »0) and containing 1.0 mol / l sodium chloride . a, 0.1 m sodium bicarbonate (pH = 8.5) removed. The amount of bound bovine serum albumin was 5 »0 mg, which corresponds to a yield of 12.5%, based on the amount of bovine serum albumin introduced into the reaction mixture.

example

It was 0.1 g of a xerogel of an acrylamide / ΙΤ, ΙίΓ-methylene-bis (acrylamide) copolymer [Akrile _x P-100 Xerogel, manufacturer: Reanal Firiomvegyszergyar, Budapest, Hungary] in 4.0 ml of a 0, 1 M Sörensen buffer solution (pH = 8.0), whereupon 1.0 ml of a 0.25 M solution of p-benzoquinone in 20% by volume aqueous dioxane was added and the suspension was added Was held at 323 2 for hours. The swollen gel was separated and sequentially

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with 70 ml, 20% by volume - aqueous dioxane and 70 ml of distilled Water washed.

The activated gel was filtered through a vacuum filter, whereupon 2.0 ml of a solution of glucose oxidase having a concentration of 20 mg / ml in a 0.1 M potassium / sodium phosphate buffer solution (pH = 7.5 ) were added. The suspension was incubated "at 277 ° 2" for 24 hours, the gel was separated and the unbound protein was washed successively with a 0.1 M sodium acetate buffer solution containing 1.0 mol / l sodium chloride (pH value = 5> 0) and. a 0.1 M sodium bicarbonate solution (pH = 8.5).

The amount of protein bound to the gel was 4.5 ms and the activity was 7.2 units / g xerogel. Such a unit corresponds to 1.0 micromoles ß-D-glucose / minute catalyzes the amount of enzyme which tion at a pH value of 7 * 0 and a temperature of 298 ⁰ K, to avoid oxidation-7th The specific activity of the bound enzyme was 8% of the specific activity of the dissolved enzyme.

Example 6

0.1 g of a xerogel of an acrylamide / ΪΓ, Ιί'- methylene-bis (acrylamide) copolymer [Akrilex P-100 Xerogel, manufacturer: Reanal Finomvegyszergyär, Budapest, Hungary] in 4.0 ml of a 0, Suspended 1 M Sörensen buffer solution (pH = = 8.0), whereupon 1.0 ml of a 0.25 M solution of p-benzoquinone in 20% by volume aqueous dioxane was added and the suspension was increased to 323 for 24 hours ° K was held. The swollen gel was burned off and washed successively with 70 ml of 20% by volume aqueous dioxane and 70 ml of distilled water.

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The activated gel was filtered through a vacuum filter and 2.0 ml of a solution of catalase having a concentration of 20 mg / ml in a 0.1 M potassium / sodium phosphate buffer solution (pH = 7.5) added. The suspension was 2Ά- hours at 277 ° incubated &", the gel was separated and the bound protein was not containing by successively washing with a 1.0 mol / l sodium chloride 0.1 m Hatriumacetatpufferlösung (pH = 5 _t 0 ) and a 0.1 M sodium bicarbonate solution (pH = 8.5).

The amount of protein bound to the gel was 5.3 mg and the activity was 2,100 units / g xerogel. Such a unit is the amount of enzyme which catalyzes at a temperature of 298 ⁰ K the decomposition of 1 micromole Wasserstoffρeroxyd / minute. The specific activity of the bound enzyme was 2% of the specific activity of the dissolved enzyme.

Example 7

0.1 g of a xerogel of an acryl amide / H ", IT-. -Methylene-bis- (acryla-mid) -Copolymers- CAkrilex P-100 Xerogel, Manufacturer: Eeanal Finomvegyszergyar, Budapest, Hungary] in 4.0 ml of a 0.1 M Sörensen buffer solution (pH value = = 8.0), whereupon 1.0 ml of a 0.25 M solution of p-benzoquinone in 20% by volume aqueous dioxane was added and the suspension was kept at 3> 23 ° K for 24 hours. The swollen gel was separated and sequentially with 70 ml of 20% by volume aqueous dioxane and 70 ml of distilled Water washed.

The activated gel was filtered through a vacuum filter, fil-

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triert and it was 2.0 ml of a 0.01 M solution of glucose in a 0.1 M potassium / sodium phosphate buffer solution (pH = 7 »5) added. The suspension was 24 hours long incubated "at 277 ° K". The gel was separated and the unbound glucose was obtained by sequential Vaschen with a 0.1 M ITa trium acetate buffer solution containing 1.0 mol / l sodium chloride (pH value = 5 * 0) and a 0.1 M sodium bicarbonate solution (pH = 8.5) removed. The amount of bound glucose was 6 micromoles, which is one Yield of 30% based on that in the reaction mixture amount of glucose introduced.

Example 8

There were JO g of a xerogel of an acrylamide / ΪΓ, ΙΓ- -Methylene-bis- (acrylamide) copolymers [Akrilex P-100 Xerogel, Manufacturer: Keanal ITinomvegyszergyar, Budapest, Hungary] in 1 200 ml of a 0.1 M potassium phosphate buffer solution or Sörensen - Pepper solution (each pH = 8) suspended, whereupon 300 ml of a 0.25 m solution of p-Bmzochiron in 20% strength. aqueous. Dioxane added The suspension was kept at 323 ° for 24 hours became. The swollen gel was separated and successively mixed with 3 liters of 20% by volume aqueous dioxane and 10 liters of distilled water washed water. The washed gel was 170-philized. There was thus obtained 24.3 S activated carrier.

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Claims (14)

Γ-Α · Λ * ϋ Claims Process for the production of immobilized compounds having 1 or more nucleophilic group (s) by activating polymeric carriers used in buffer solutions with activating or coupling agents used in solution and treating the compounds having 1 or more nucleophilic group (s) with the polymeric carriers activated in this way and using separation and washing operations, characterized in that the polymer / carrier used is a polymer containing amide groups in a buffer solution with a pH of 3 "to 11 that is free from amino, sulfhydryl and / or substances containing hydroxyl groups swells, according to whether a solution of a quinone in a water-miscible organic solvent is added in such an amount that the final concentration of the quinone is 2 to 100 MilUmoVl; then the mixture is 0.5 " activated for up to 4-8 hours at 273 to 34-3 ° K, then the unreacted quinone by washing mi t a mixture of water and a water-miscible organic solvent and then removed with distilled water and / or a buffer solution, then a 0.001 to 1.0 m buffer solution with a pH of 3 to from the resulting gel containing deactivated amide groups 11 of the 1 or more nucleophilic group (s) containing compounds) with low molecular weight or a buffer solution of the 1 or more nucleophilic group (s) containing macromolecular compound (s) with a pH of 3 to 11 from a concentration of 3 to 100 ηg / ml is added, the suspension obtained is incubated at 260 to 313 ° K for 20 to 30 hours and d. ··; oh the gel r-'o burns and the COPY - A- unbound (η) 1 or more nucleophilic group (s) separating (s) comprising compound (s) by washing. 2.) Method according to claim 1, characterized in that that one has as a buffer solution (s) for the amide groups Polymer and / or the compound (s) having 1 or more nucleophilic group (s) with low Molecular weight or macromolecular (s) Compound (s) those having a pH of 6 to 8 are used. 3.) Process according to claim 1 or 2, characterized in that the quinone used is p-benzoquinone. 4.) The method according to claim 1 to 3 »characterized in that the solution of the quinones in one such an amount is used that its final concentration is 40 to 60 millimoles / l. 5-) The method of claim 1 to 4, characterized in that up to 33O ⁰ K performs the activation of the mixture of the buffer solution of the amide groups serving as carrier for facing the polymer and the solution of the Chinones at 310th 6.) The method according to claim 1 to 5 »characterized in that the activation of the mixture from the Buffer solution of the polymer containing amide groups and the solution of the quinone Performs for 20 to JO hours. 7.) The method according to claim 1 to 6, characterized in that the incubation of the suspension obtained the low molecular weight compound (cn) having 1 or more nucleophilic group (s) COPY . 3- or macromolecular compound (s) and the activated amide group serving as a carrier Polymers carried out at 260 to 280 ° K. 8.) Method according to claim 1 to 7, characterized in that that the incubation of the Sus-. pension of 1 or more nucleophilic group (s) Compound (s) with low molecular weight or macromolecular compound (s) .and ■ the activated amide groups serving as a carrier on- ■■ · '■■ pointing polymer for 22 to 26 hours. 9.) Activated polymeric carriers, suitable for immobilizing 1 or more nucleophilic group (s) containing compound (s), which are obtained by activating polymeric carriers used in buffer solutions with activating or coupling agents used in solution and using separating and washing operations have been obtained, characterized in that for their preparation, a polymer containing amide groups has been swollen in a buffer solution with a pH of 3 to 11 which is free from substances containing amino, sulfhydryl and / or hydroxyl groups as the polymeric carrier , then the solution of a quinone in a water-miscible organic solvent in such an amount that the final concentration of the quinone is 2 to • 100 MiUiscO / l, then the mixture for 0.5 to 48 hours at 273 to 343 ⁰ K activated and then the not unoccupied quinone by washing with a mixture of water and e has been removed in a water-miscible organic solvent and then with distilled water and / or a buffer solution. ; ■ *. ■ -. COPY 10.) Carrier according to claim 9 »characterized in that as a buffer solution for the amide groups Polymer one having a pH of 6 to 8 has been used. 11.) Carrier according to claim 9 or 10, characterized in that p-benzoquinone is used as the quinone has been. 12.) Carrier according to claim 9 "to 11, characterized in that that the quinone solution has been used in such an amount that its final concentration 40 to 60 millimoles / l. 13 ·) Carrier according to claim 9 to 12, characterized in that that the activation of the mixture from the buffer solution of the carrier containing amide groups Polymer and the solution of quinones at 310-330 K. 14.) Carrier according to claim 9 to 13, characterized in that the activation of the mixture of the buffer solution of the polymer containing amdi groups and the solution of the quinone for 20 to 30 hours has been carried out for a long time. description